ARTICLE pubs.acs.org/jpr
Proteomic Characterization of Human Milk Whey Proteins during a Twelve-Month Lactation Period Yalin Liao,† Rudy Alvarado,‡ Brett Phinney,‡ and Bo L€onnerdal*,† †
Department of Nutrition, ‡Genome Center Proteomics Core Facility, University of California, Davis, California 95616, United States ABSTRACT: Human milk is a rich source of bioactive proteins that support the early growth and development of the newborn. Although the major components of the protein fraction in human milk have been studied, the expression and relative abundance of minor components have received limited attention. We examined the expression of low-abundance proteins in the whey fraction of human milk and their dynamic changes over a twelve-month lactation period. The low-abundance proteins were enriched by ProteoMiner beads, and protein identification was performed by liquid chromatography tandem mass spectrometry. One hundred and fifteen proteins were identified, thirty-eight of which have not been previously reported in human colostrum or milk. We also for the first time described differences in protein patterns among the low-abundance proteins during lactation. These results enhance our knowledge about the complexity of the human milk proteome, which constitutes part of the advantages to the breastfed infant. KEYWORDS: human milk, whey, protein, proteomics, LC-MS/MS
’ INTRODUCTION Milk is the most important food for the newborn, because of its unique nutrient composition, immune components, anti-infective factors, and metabolic enzymes, which all contribute to meet the critical needs for growth and development during early life. Several studies have shown that breast-feeding is associated with a lower incidence of obesity, diabetes, and cardiovascular disease later in life.1-4 The protein compartment of milk plays a critical role in achieving many of the beneficial outcomes of breast-feeding. Human milk is unique in that the whey fraction contains the major part of protein (60-80%),5 whereas casein is a smaller fraction. Minor proportions of proteins are present in the other fractions of cells and milk fat globule membranes (MFGM) (see reviews6-8). Protein intake and composition are quite different in formula-fed infants and breast-fed infants. Human milk contains bioactive human milk proteins that are likely to have significant short-term and long-term beneficial effects.9-11 A comprehensive understanding of the human milk protein profile is expected to contribute not only to our understanding of milk biogenesis and functions provided to the newborn but may also provide guidance on how to develop infant formulas more similar in protein composition to human milk. Proteomics technologies have greatly advanced our in-depth knowledge on milk proteins (see reviews12,13). Previous milk proteomics studies included characterization of host defense proteins14 and N-linked glycoproteins.15 Significant efforts have also been made to characterize the MFGM proteins.16,17 In terms r 2011 American Chemical Society
of the identification of whey proteins, Murakami et al.18 used 2-DE and microsequencing and identified 22 well-resolved proteins out of 400 protein spots on two-dimensional (2D) electrophoresis. By improving 2D liquid chromatography tandem mass spectrometry (LC-MS/MS), Palmer et al.19 were able to identify 152 proteins in mature human milk whey. The dynamic changes in milk proteins during lactation, however, have not been comprehensively examined. Human colostrum and mature milk have been found to differ in the quantity of total protein as well as in composition.20 The objective of this study was to examine the protein profile of human milk over a twelve-month lactation period and to identify proteins with differentially regulated abundance during lactation.
’ EXPERIMENTAL PROCEDURES Milk Sample Collection
This study procedure was approved by the Institutional Review Board (IRB) at University of California Davis. Colostrum and milk samples from 1, 2, 3, 6, and 12 months of lactation were collected from one breast (at least 2-4 h after prior nursing) by manual expression (colostrum) or manual breast pump into 50 mL polypropylene containers. Mothers who delivered singleton term infants (gestational age 38-42 weeks by maternal dates of Received: October 12, 2010 Published: March 01, 2011 1746
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and treated with trypsin (modified trypsin, sequencing grade, Promega) at a 1:50 enzyme-to-substrate ratio (w/w) overnight at 37 °C. The trypsin digested samples were dried and redissolved in 2%ACN/0.1%TFA for MS analysis. LC-MS/MS
Figure 1. SDS-PAGE analysis of a human milk whey sample (2 months of lactation) before and after Proteominer treatment. Ten milligrams of total whey protein was applied onto the Proteominer beads. Ten micrograms of total proteins were run through an 8% SDS-PAGE, and the gel was stained with Coomassie Brilliant Blue. Lane 1, human milk whey before Proteominer treatment; lane 2, human milk whey after Proteominer treatment. The relative abundance of minor proteins in milk was enhanced, and arrows indicated enriched protein clusters. MWM: molecular weight marker.
best obstetric estimate) were recruited, and mothers with illnesses, such as cold, mastitis, and flu were excluded. All women were exclusively breast feeding up to 6 months and partially up to 12 months of lactation. There are five samples collected at each time point. Colostrum was collected within 48 h of lactation initiation,21 and all other samples were consistently collected between 8 and 10 a. m. Samples were immediately stored at -20 °C until further analysis. Protein Extraction
Milk samples (5 mL) were thawed at 4 °C. CaCl2 was added to adjust the final calcium concentration to 0.06 M. The pH was adjusted to 4.6 and samples were incubated at room temperature for 1 h.22 The samples were then subjected to centrifugation at 13 000g for 30 min twice. The infranatant whey fraction was collected. Protease inhibitors (Roche) were added to the whey. The minor proteins in the whey fraction were enriched by the ProteoMiner kit (Bio-Rad) according to the manufacturer’s recommendations, and 10 mg of whey proteins were used for each sample. The ProteoMiner bead consists of a unique library of hexapeptides, which bind proteins of different abundance with same capacity; the fast saturation of the high-abundance proteins allows the enrichment of medium- to low-abundance proteins.23 For whey extracted from milk of each subject, two samples (treated or untreated by ProteoMiner bead) were derived, and data from the two samples were combined. Final concentrations of the whey proteins were measured by the Microplate BCA protein assay kit (Fisher Scientific). In-solution Digestion
Soluble proteins were digested in-solution using a standard Trypsin digestion protocol. Briefly, the proteins were dried in a vacuum centrifuge and then resuspended in 50 mM ammonium bicarbonate. The proteins were then reduced with tris(2-carboxyethyl)phosphine (TCEP) (Pierce), alkylated by iodoacetamide,
A Paradigm MG4 HPLC System (Michrom Bio Resources) coupled with a Thermo LTQ ion trap mass spectrometer (Thermo Scientific) through a Michrom Advance Captivespray source was used for protein separation and analysis. Fifteen micrograms of each digested sample were loaded onto a trap column (Zorbax 300SB-C18, 5 μm, 0.3 mm 5 mm, Agilent Technologies Inc.) and desalted online. Peptides were then eluted from the trap and separated with a reverse-phase Michrom Magic C18AQ (200 μm 150 mm) capillary column at a flow rate of 2 μL/min. Peptides were eluted using a 90 min gradient of 2-35% B over 60 min, 35-80% B for 15 min, held at 80% B for 1 min, 80%-5% B in 1 min, and re-equilibrated for 13 min at 5% B (A = 0.1% formic acid, B = 100% Acetonitrile) and directly sprayed into the mass spectrometer. The mass spectrometer was operated using a standard top 10 method, where one survey scan was followed by 10 MS/MS scans of the most intense ions eluting from the column. Dynamic exclusion was enabled. Immunoblotting Analysis
Whey proteins from each group were pooled, and 20 μg were electrophoresed through 8% polyacrylamide gel, transferred onto nitrocellulose membrane at 350 mA for 60 min, and blocked overnight in 1 PBS/0.1% Tween-20 (PBST) with 5% BSA at 4 °C. Antibodies against xanthine oxidase, transcobalamin I, and perilipin-2 were purchased from Santa Cruz Biotechnologies. Bands were detected using Super Signal Femto chemiluminescent reagent (Pierce) and quantified using the Chemi-doc gel quantification system (Bio-Rad). All data were normalized to β-actin.
Data Analysis
Database Searching. Tandem mass spectra were extracted by BioWorks version 3.3. Charge state deconvolution and deisotoping were not performed. All MS/MS samples were analyzed using X! Tandem (www.thegpm.org; version TORNADO (2010.01.01.4). X! Tandem was set up to search the Uniprot human complete proteome set database (2010_07, 21,525 entries) and 110 nonhuman common laboratory contaminants from the common repository of adventitous proteins database (www.thegpm.org) plus an equal number of reverse sequences assuming the digestion enzyme trypsin. X! Tandem was searched with a fragment ion mass tolerance of 0.40 Da and a parent ion tolerance of 1.8 Da. Iodoacetamide derivative of cysteine was specified in X! Tandem as a fixed modification. Deamidation of asparagine and glutamine, oxidation of methionine and tryptophan, Sulphone of methionine, tryptophan oxidation to formylkynurenin of tryptophan and acetylation of the n-terminus were specified in X! Tandem as variable modifications. Criteria for Protein Identification. Scaffold (version Scaffold_3_00_03, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 90.0% probability as specified by the Peptide Prophet algorithm.24 Protein identifications were accepted if they could be established at greater than 95.0% probability and contained at least two identified peptides. Protein probabilities 1747
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Table 1. List of Identified Proteins from Human Milk Wheya serial no.
identified proteins
UniProt
molecular
no. of unique
sequence
accession no.
weight (kDa)
peptides
coverage (%)
1b
14-3-3 protein zeta/delta
P63104
28
2
18
2b
60S acidic ribosomal protein P1
P05386
12
2
42
3b
78 kDa glucose-regulated protein
P11021
78
6
12
4
actin, cytoplasmic 1
P60709
42
7
31
5b
alcohol dehydrogenase [NADPþ]
P14550
37
2
6
R-1-acid glycoprotein 1
P02763
24
5
28
7 8
R-1-antichymotrypsin R-1-antitrypsin
P01011 P01009
48 47
15 11
43 36
9
R-1B-glycoprotein
P04217
54
3
16
10
R-2-HS-glycoprotein
P02765
39
2
13
11
R-amylase 1
P04745
58
5
18
12b
R-enolase
P06733
47
7
27
13
R-lactalbumin
P00709
16
21
79
14
R-S1-casein
P47710
22
21
83
15 16
amyloid β A4 protein apolipoprotein A-I
P05067 P02647
87 31
2 8
4.3 30
17b
apolipoprotein A-II
P02652
11
2
28
18
apolipoprotein D
P05090
21
3
21
19
β-1,4-galactosyltransferase 1
P15291
44
6
28
20
β-2-microglobulin
P61769
14
6
41
7.4
21b
β-actin-like protein 2
Q562R1
42
2
17
22
β-casein
P05814
25
51
86
23 24
bile salt-activated lipase, carboxyl ester lipase butyrophilin subfamily 1 member A1
P19835 Q13410
79 59
32 15
40 37
25
C4b-binding protein R chain
P04003
67
5
13
26b
calmodulin
P62158
17
2
45
27
calreticulin
P27797
48
7
26
28
carbonic anhydrase 6
P23280
35
8
44
29
ceruloplasmin
P00450
122
3
30
chitinase-3-like protein 1
P36222
43
6
23
31b 32
chordin-like protein 2 clusterin
Q6WN34 P10909
47 52
11 20
37 42
33b
cofilin-1
P23528
19
2
17
34
complement C3
P01024
187
33
26
35
complement C4-B
P0C0L5
193
33
28
36b
copine-5
Q9HCH3
66
2
37
cystatin-C
P01034
16
2
19
38b
fatty acid synthase
P49327
273
18
12
39 40
fatty acid-binding protein, heart fibrinogen γ chain
P05413 P02679
15 52
10 3
77 8.8
41b
fibroblast growth factor-binding protein 1
Q14512
26
2
13
42b
folate receptor R
P15328
30
2
12
43b
fructose-bisphosphate aldolase A
P04075
39
2
44
galectin-3-binding protein
Q08380
65
12
29
45b
gelsolin
P06396
86
6
10
46
glyceraldehyde-3-phosphate dehydrogenase
P04406
36
3
13
47b 48
golgi-associated plant pathogenesis-related protein 1 haptoglobin
Q9H4G4 P00738
17 45
2 8
17 22
49b
hemoglobin subunit delta
P02042
16
2
16
50b
histone H2B type 1-D
P58876
14
2
15
51
Ig R-1 chain C region
P01876
38
21
70
52
Ig R-2 chain C region
P01877
37
4
63
53
Ig γ-1 chain C region
P01857
36
8
35
1748
5.4
6.9
7.1
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Table 1. Continued serial no.
identified proteins
UniProt
molecular
no. of unique
sequence
accession no.
weight (kDa)
peptides
coverage (%)
54
Ig heavy chain V-I region HG3
P01743
13
2
20
55
Ig heavy chain V-III region BRO
P01766
13
2
25
70
Ig J chain
P01591
18
9
37
56
Ig κ chain C region
P01834
12
13
100
57
Ig κ chain V-I region AG
P01593
12
2
31
58
Ig κ chain V-II region TEW
P01617
12
2
18
59
Ig κ chain V-III region SIE
P01620
12
3
46
60 61
Ig κ chain V-III region VG (Fragment) Ig κ chain V-IV region (Fragment)
P04433 P06312
13 13
2 2
36 30
62
Ig λ chain V-I region HA
P01700
12
2
23
63
Ig λ chain V-III region LOI
P80748
12
3
31
64
Ig λ chain V-III region SH
P01714
11
2
25
65
Ig λ chain V-IV region Hil
P01717
12
2
18
66c
Ig λ-1 chain C regions
P0CG04
11
2
93
67c
Ig λ-2 chain C regions
P0CG05
11
12
96
68c 69
Ig λ-7 chain C region Ig μ chain C region
A0M8Q6 P01871
11 49
4 8
80 28
71
κ-casein
P07498
20
21
60
72
lactadherin
Q08431
43
19
76
73
lactoferrin
P02788
78
121
91
74
leucine-rich R-2-glycoprotein
P02750
38
6
27
75
lipoprotein lipase
P06858
53
6
23
76b
L-lactate
77b 78
lysine-specific demethylase 5A lysozyme C
79b 80
dehydrogenase B chain
P07195
37
2
P29375 P61626
192 17
2 6
macrophage mannose receptor 1
P22897
166
25
23
monocyte differentiation antigen CD14
P08571
40
9
41
81
mucin-1
P15941
122
2
82
mucin-16
Q8WXI7
2353
2
0.19
83
mucin-4
Q99102
232
2
1.6
84
osteopontin
P10451
35
7
23
85 86b
peptidyl-prolyl cis-trans isomerase A peptidyl-prolyl cis-trans isomerase B
P62937 P23284
18 24
5 2
52 10
87b
perilipin-2
Q99541
48
7
23
88b
perilipin-3
O60664
47
3
12
89b
phosphatidylethanolamine-binding protein 1
P30086
21
3
37
90
polymeric immunoglobulin receptor
P01833
83
38
51
91
proactivator polypeptide
P07602
58
8
20
92b
probable E3 ubiquitin-protein ligase MYCBP2
O75592
510
2
93 94
pro-epidermal growth factor prolactin-inducible protein
P01133 P12273
134 17
3 5
3.5 44
95b
protein disulfide-isomerase A1
P07237
57
8
22
96b
protein disulfide-isomerase A4
P13667
73
2
3.7
97b
protein piccolo
Q9Y6 V0
567
2
1.2
98b
protein S100-A8
P05109
11
2
99b
rab GDP dissociation inhibitor β
P50395
51
2
6.7
100b
retrotransposon-like protein 1
A6NKG5
155
2
4
101b 102b
RNA-binding protein with serine-rich domain 1 sclerostin domain-containing protein 1
Q15287 Q6 4U4
34 23
2 3
18 17
103
serum transferrin
P02787
77
4
104
serum albumin
P02768
69
68
105b
sulfhydryl oxidase 1
O00391
83
7
13
106b
tenascin
P24821
241
70
39
1749
7.8 3 52
1.8
0.86
24
8.3 84
dx.doi.org/10.1021/pr101028k |J. Proteome Res. 2011, 10, 1746–1754
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Table 1. Continued serial no.
identified proteins
UniProt
molecular
no. of unique
sequence
accession no.
weight (kDa)
peptides
coverage (%)
107
thrombospondin-1
P07996
129
5
108
transcobalamin-1
P20061
48
2
4.9
109
transthyretin
P02766
16
2
24 26
7.9
110
triosephosphate isomerase
P60174
27
4
111b
UTP--glucose-1-phosphate uridylyltransferase
Q16851
57
2
112
vitamin D-binding protein
P02774
53
5
113
vitronectin
P04004
54
4
13
114 115
xanthine dehydrogenase/oxidase zinc-R-2-glycoprotein
P47989 P25311
146 34
48 12
40 46
6.3 16
a The complete identification statistics can be found in the Scaffold located in the proteome data set uploaded to the Tranche proteome commons repository (Experimental Procedures). b Indicates proteins not identified from human milk whey previously. c Indicates proteins that compose a group and are not distinguished using the current data set.
Figure 2. Pie graph representation of functional characteristics of human milk whey proteins. Abbreviations used: C;S (cell communication;signal transduction); N (nucleobase, nucleoside, nucleotide and nucleic acid regulation); I (immune response); G;M (cell growth and/or maintenance); T (transport); P (protein metabolism); M;E (metabolism;energy pathways); M (multiple).
were assigned by the Protein Prophet algorithm.25 Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony; in this study, these proteins are those with accession numbers A0M8Q6, P0CG04, and P0CG05. Using these Scaffold criteria a False discovery rate was calculated as 0.2% on the peptide level and 8.1% on the protein level according to Decoy/Target as discussed in ref 26. Spectral Counting and Shared Peptide Refinement. Label free quantitation was performed using a standard spectral counting method. Scaffold 3.3 was used to sum spectral counts and group peptides into proteins. An in house script was written to refine spectral counts from peptides shared across multiple proteins according to the method in.27 The refined spectral counting data was filtered so a protein required a minimum of 4 spectral counts in any one category and a heatmap was generated using the Hierarchical clustering tool in Spotfire 3.2 using an Unweighted Pair-Group Method with Arithmetic mean (UPGMA) method. The most differentiated proteins were identified by calculating a P value using a One-Way ANOVA analysis in Spotfire 3.2. Proteomics Data Set. The data associated with this manuscript may be downloaded from ProteomeCommons.org Tranche using the following hash: 3u3rLRMef0 4TXhg1YbARPGa1zS3lf34K6hKm9xAQqCHQeGuuBZKl56tO4RgK7IP3PGOdfk/ 0VV/Ga8LVub6tvL2sFgAAAAAAAAwrg==
Figure 3. Pie graph representation of subcellular localization of human milk whey proteins.
The hash may be used to prove exactly what files were published as part of this manuscript’s data set, and the hash may also be used to check that the data has not changed since publication.
’ RESULTS Human Milk Whey Extraction and Enrichment of Minor Whey Proteins
The aqueous whey fraction of human milk was isolated by removing the fat layer and casein precipitate after extensive centrifugation. A protein with a molecular weight of ∼78 kDa (lactoferrin) predominates in the whey; however, after Proteominer treatment, the relative abundance of the ∼78 kDa band is decreased, and the abundance of minor proteins was increased. The Proteominer beads had little effect on remaining casein residues in the whey, as indicated by the bands around 25 kDa in Figure 1. Characterization of Human Milk Whey Proteins
To extensively characterize human milk whey proteins, the identified proteins from all the individual samples were pooled, which resulted in a total of 115 proteins (Table 1). These proteins were categorized according to their functions or subcellular distribution assigned in the Human Protein Reference Database (http:// www.hprd.org/) and UniProt Database release 15.14 (http:// www.uniprot.org/). Eight functional groups were noted (Figure 2): 17.4% of all proteins are involved in cell communication;signal transduction, 2.6% in nucleobase, nucleoside, nucleotide and nucleic acid regulation; 34.8% in immune response, 5.2% in cell growth and/or maintenance, 12.2% in general transport, 7.8% in protein metabolism, including protein translation, folding, trafficking, and so forth, 16.5% in metabolism;energy production 1750
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Figure 4. Heat map presentation of spectral counting data. Hierarchical clustering analysis was performed using Euclidian distance. Each column represents the relative spectral counting of each group. Colors represent the scaled fold-change of spectrum counts between samples within a row. Red color represents higher expression and blue color represents lower expression. Group 1, colostrum; group 2, 1 month lactation; group 3, 2 months lactation; group 4, 3 months lactation; group 5, 6 months lactation; group 6, 12 months lactation.
pathways, and 3.5% are typical multifunctional proteins. The proteins were also divided into five groups according to their subcellular distributions (Figure 3): 23.5% are cytoplasmic proteins, 55.7% are extracellular proteins, 2.6% are nucleus proteins, 11.3% are cellular membrane proteins, and 7.0% are located at multiple subcellular localizations. Relative Expression Level of Human Milk Whey Proteins during Lactation
Spectral counting and differentiation expression analysis indicated that milk whey proteins are expressed with different abundances during the course of lactation, as can be seen in Figures 4 and 5. Proteins with more than four spectral counts were included in the heatmap. Each column represents the average spectral counts of each group, each row represents individual protein. Proteins such as R-1-antitrypsin, carbonic anhydrase, chordin-like protein 2, galectin-3-binding protein, lactadherin, lipoprotein lipase, and tenascin are expressed at higher concentrations during early than during late lactation; proteins such as fatty-acid binding protein, lysozyme C, monocyte differentiation antigen, proactivator polypeptide, transcobalamin-1, and zinc-R-2-glycoprotein are expressed in higher abundance after 6 months lactation. To validate our spectral counting method and show that similar results are obtained with conventional semiquantitative methods, immunoblotting analyses were performed for selected proteins. Figure 6 shows the immunoblotting confirmation for the presence and relative abundance of xanthine oxidase, transcobalamin I, and perilipin-2.
’ DISCUSSION In this study, the human milk whey proteome was identified by liquid chromatography tandem mass spectrometry (LC-MS/MS)based analysis. One hundred and fifteen proteins were identified in human milk whey fraction, thirty-eight of which have previously not been reported in human milk. Their dynamic expression patterns during a 12 month lactation period are also described. Minor proteins in human milk whey were enriched by Proteominer Protein Enrichment technology (Bio-Rad) to increase the number of unique peptides and proteins detectable by LCMS/MS. Our data showed that the Proteominer technique is a valid approach to increase the odds of detecting proteins of very low abundance. It should be noted that although minor proteins were enriched, major whey proteins (e.g., lactoferrin and Rlactalbumin) were still detected in the whey fraction. Furthermore, casein subunits, in principle belonging to the casein fraction but most likely representing unassembled casein micelles, were also detected. Some milk fat globule membrane proteins (e.g., mucin-4) were also found, most likely representing a small fraction dissociated from the membranes. Proteins involved in growth/maintenance and immunity support comprise 40.0% of the total proteins, representing key functions of milk whey proteins. Newly identified growth promoting proteins include gelsolin (actin-binding protein), cofilin-1 (actin-binding), β-actin-like protein 2, chordin-like protein 2, and retrotransposon-like protein 1. It should be noted that chordin-like protein 2 is a secreted protein expressed in retina, 1751
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Figure 6. Immunoblotting analysis for relative expression of human milk whey proteins. Xanthine oxidase, transcobalamin I, and perilipin-2 were analyzed in human whey extracted from colostrum and milk from 1, 2, 3, 6, and 12 months of lactation. β-actin served as internal control. Values are means ( SEM run in triplicates, letters indicate significant differences between groups (P < 0.05).
Figure 5. Box plot of protein differential expression of xanthine oxidase, perilipin-2, tenascin, β-casein, and chordin-like protein 2. Each graph represents the median spectral counts and standard error of individual replicate. There are five replicates for each protein.
involved in regulating angiogenic homeostasis by inhibiting the antiangiogenic factor bone morphogenic protein 4 (BMP-4).28 A group of 40 proteins were identified to function as immune
modulators. Among these proteins, golgi-associated plant pathogenesis-related protein 1 and protein S100-A8 have not been identified previously in human milk. Golgi-associated plant pathogenesis-related protein 1 localizes to lipid-enriched microdomains in the Golgi complex and is the first member of PR-1 superfamily without signal sequence, resulting in cytoplasmic localization, and is associated with microdomains;29 protein S100-A8 is a calcium binding protein that has antimicrobial activities and serves as a proinflammatory mediator in acute and 1752
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Journal of Proteome Research chronic inflammation by regulating NFκB-dependent genes.30 According to the label-free spectral counting data, all the immune factors are produced throughout the 12 month lactation period, further supporting the potential immunological benefits in transferring maternal immunity and helping the newborn elicit effective immune responses to microorganisms. It is known that immune function is different in breast-fed and formula-fed infants,31 and it is possible that some components of this group may be in part responsible for these differences. Dietary fat intake during infancy is very high, because of their high energy requirement and beneficial effects of fat on growth and development of the brain and vision. Approximately 50% of the total energy intake is acquired from milk lipids during the first month after birth, and 35% of an infant’s weight gain during the first 6 months consists of body fat.32,33 In the present study, 13 proteins were identified to be involved in lipid metabolism: apolipoproteins A-I, A-II, and D, butyrophilin subfamily 1 member A1, carboxyl ester lipase (bile salt activated lipase), fatty acid-binding protein, fatty acid synthase, lipoprotein lipase, perilipin-2, perilipin-3, triosephosphate isomerase, zinc-R2-glycoprotein, and xanthine dehydrogenase. While lipases in the milk contribute to effective digestion and retention of milk fat, the apolipoproteins function to transport fatty acids in the circulation. Perilipin-2/3, fatty acid synthase, and triosephosphate isomerase were for the first time identified in human milk. Perilipin-2, also known as lipid droplet-associated protein, is an important regulator of lipid storage, and functions as a protective coating of lipid droplets in adipocytes to prevent lipolysis;34 fatty acid synthase is an indispensible component of lipogenesis and the energy production pathway.35 The physiological function of triosephosphate isomerase is to maintain the homeostasis between dihydroxyacetone phosphate and glyceraldehyde-3-phosphate produced by aldolase in glycolysis, which is interconnected to the pentose phosphate pathway and to lipid metabolism via triosephosphates.36 Thus, milk contains enzymes that help to balance lipid synthesis and breakdown. Some of these enzymes, for example carboxyl ester lipase (bile salt activated lipase), are known to be active in lipid digestion in the gut of the breast-fed newborn,32 but it is quite likely that some of these enzymes also reflect metabolic activities in the mammary gland (milk biogenesis). In addition, several proteins involved in energy production were identified, including proactivator polypeptide, alcohol dehydrogenase [NADPþ], R-enolase, fructose-bisphosphate aldolase A, L-lactate dehydrogenase B chain, and UTPglucose-1-phosphate uridylyltransferase, most of which are involved in glycolysis of the energy production pathway. An interesting group of proteins are involved in regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolism. None of the three proteins have been identified in human milk previously. They are involved in maintenance of nucleosome structure of the chromosomal fiber in eukaryotes (Histone H2B type 1-D), RNA splicing, mRNA processing (RNA-binding protein with serine-rich domain 1), and transcription regulation (lysine-specific histone demethylase 1A). Where these activities are exerted is not known, but they may also be involved in mammary gland metabolism. Proteins involved in protein metabolism machineries are also a significant part of the identified proteins, comprising 7.8% of total proteins. For example, peptidyl-prolyl cis-trans isomerases A and B are involved in protein folding; protein disulfideisomerase A1 and A4 are involved in facilitating protein
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secretion; cystatin C is an inhibitor of cysteine proteinases; and 60S acidic ribosomal protein P1 functions in translational elongation. Label free spectral counting was used to describe the relative quantification of minor milk proteins during the course of lactation. Among the minor proteins, we identified the following proteins having significantly higher expression levels in colostrum and 1 month milk whey: R-1-antitrypsin, R-lactalbumin, carbonic anhydrase 6, chordin-like protein 2, galectin-3-binding protein, lactadherin, lactoferrin, prolactin-inducible protein, and tenascin. Most of these proteins are involved in immunestimulating functions and it is possible that their functions are more important during the newborn period. Immunoblotting analysis was performed to verify the spectral counting data, and the obtained immunoblotting data for xanthine oxidase, transcobalamin I, and perilipin-2 all mimic the spectral counting data, providing support for using the spectral counting approach to quantify relative changes in milk protein abundances. In summary, the present study was the first attempt to comprehensively address the human milk protein profile and changes in the relative abundance of the proteins during a twelvemonth lactation period. Knowledge obtained from the results will enhance our understanding of the complexity of the human milk proteome, provide insights into proteins involved in milk biogenesis and, possibly, into proteins having bioactivity in the recipient breast-fed infant and thus contributing to suggestions on how to develop infant formulas closer to human milk.
’ CONCLUSIONS There has been previous research that used proteomics methods to identify human milk proteins. Our study not only identified known and novel proteins present in human milk but also revealed the relative expression pattern of each individual protein along the course of 12 months of lactation. Furthermore, immunoblotting analyses were used to validate the human milk proteins. In summary, this study is the first attempt to use quantitative proteomic approach, which enables a more comprehensive understanding of human milk proteome.
’ AUTHOR INFORMATION Corresponding Author
*Address: Department of Nutrition, University of California, Davis, One Shields Ave., Davis, CA 95616. Tel.: þ1 530-7528347. Fax: þ1 530-752-3564. E-mail:
[email protected].
’ ACKNOWLEDGMENT The authors would like to thank Dr. Deshanie Rai for constructive discussions and Mead Johnson Nutrition for supporting this study. We also want to thank Dr. Ian J. Griffin for milk sample collections; Tim Wehr from BioRad for supplying the ProteoMiner kits; Xiaogu Du for milk whey extraction; and Diana Tran and Bonnie Ching for MS sample preparations. ’ REFERENCES (1) Martin, R. M.; Gunnell, D.; Smith, G. D. Breastfeeding in infancy and blood pressure in later life: systematic review and meta-analysis. Am. J. Epidemiol. 2005, 161, 15–26. (2) Koletzko, B.; von Kries, R.; Monasterolo, R. C.; Subias, J. E.; et al. Infant feeding and later obesity risk. Adv. Exp. Med. Biol. 2009, 646, 15–29. 1753
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